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First published online 27 January 2009
doi: 10.1242/jcs.033464

Journal of Cell Science 122, 524-534 (2009)
Published by The Company of Biologists 2009
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Drebrin A regulates dendritic spine plasticity and synaptic function in mature cultured hippocampal neurons

Anton Ivanov1, Monique Esclapez2, Christophe Pellegrino1, Tomoaki Shirao3 and Lotfi Ferhat1,4,*

1 INMED/INSERM U29, Parc Scientifique de Luminy, 13273, Marseille, France
2 INSERM U 751, Université d'Aix-Marseille, Hôpital de la Timone, Marseille, France
3 Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan
4 CNRS UMR 6184, Neurobiologie des Interactions Cellulaires et Neurophysiopathologie (NICN), IFR Jean Roche, Marseille, F-13020, France


Figure 1
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  		Fig. 1. Effects of DA-GFP overexpression on spine morphology and network activity. Cultured neurons were cotransfected on DIV 21 with RFP (A) and GFP (A'), RFP (B) and DA-GFP (B'), RFP (C) and GFP-DA{Delta}ABS (C'), or RFP (D) and GFP-ABS (D'). At 23 DIV, neurons were fixed and then analyzed. RFP channel is shown in all panels to outline dendritic morphology. (A'',B'',C'' and D'') Merged images. Scale bar: 10 µm. (E-G) Quantification of DA-GFP effects on protrusion plasticity. Histograms showing the average length (E), width (F) and density (G) of protrusions of GFP, DA-GFP, GFP-DA{Delta}ABS and GFP-ABS neurons. As with GFP spines (H-H''), the long spines induced by DA-GFP (I-I'') were associated with functional excitatory synaptic contacts. GFP (H) or DA-GFP (I) neurons immunostained for synaptophysin (H' and I'). (H'' and I'') Merged images. Some synaptophysin clusters are not in close apposition to the spines of GFP or DA-GFP neurons because they are probably opposed to dendritic shafts or spines of nontransfected neurons. Scale bar: 10 µm. *P<0.05, **P<0.01, ***P<0.001, Bonferroni's test. (J) Examples of sPSCs recorded at –60 mV in GFP (black trace) and DA-GFP neurons (gray trace). (K) Histogram showing the sPSCs charge transfer in all recorded GFP and DA-GFP neurons. All data in E-G and K are mean ± s.e.m.

 

Figure 2
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  		Fig. 2. Effects of DA-GFP overexpression on glutamate function. Mixed hippocampal cultures were transfected at 21 DIV with GFP or DA-GFP, and 1 day later, the transfected neurons were recorded. Electrophysiological recordings of GFP neurons used as controls were compared with those of DA-GFP neurons. (A) Examples of mEPSCs recordings from GFP (black trace) and DA-GFP neurons (gray trace). (B,C) Cumulative probability plots of amplitude and frequency of mEPSCs in GFP and DA-GFP neurons. The average amplitude and frequency of mEPSCs in GFP and DA-GFP neurons are shown as insets. *P<0.05, **P<0.01, K-S test. (D-F) Histograms showing the average rise time, decay time, and charge transfer of mEPSCs respectively in GFP and DA-GFP neurons. Traces in D and E show the rise and decay time, respectively, of the average mEPSCs. **P<0.01, U-test.

 

Figure 3
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  		Fig. 3. Effects of DA-GFP overexpression on GABA function. Mixed hippocampal cultures were transfected at 21 DIV with GFP or DA-GFP, and 1 day later, the transfected neurons were recorded. Electrophysiological recordings of GFP neurons used as controls were compared with those of DA-GFP neurons. (A) Examples of mIPSCs recordings from GFP neurons (black trace) and DA-GFP neurons (gray trace). (B,C) Cumulative probability plots of amplitude and frequency of mIPSCs in GFP and DA-GFP neurons. The average amplitude (B) and frequency of mIPSCs (C) in GFP and DA-GFP neurons are shown in insets. **P<0.01, ***P<0.001, K-S test. (D-G) Histograms showing the average rise time, decay time, charge transfer and inhibition to excitation transfer charge ratio of mEPSCs, respectively, in GFP and DA-GFP neurons. *P<0.05, t-test; **P<0.01, U-test.

 

Figure 4
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  		Fig. 4. Effects of DA-GFP on the density of GABA and glutamate receptors and synaptic proteins. Transfected neurons were recorded in whole-cell configuration at –60 mV for GABAA- or AMPA-generated responses and –20 mV for NMDA-mediated current in the presence of TTX and strychnine. (A) Example of current responses induced by bath application of receptor agonists, Isoguvacine (20 µM), AMPA (100 µM) and NMDA (50 µM, with 10 µM glycine, NMDA coactivator) to activate GABAA, AMPA and NMDA receptors, respectively, 10 seconds after starting perfusion (double arrows) with agonist-containing solution. (B) Quantitative analysis revealed that DA overexpression did not modify significantly the average current density of the agonist-mediated responses compared with that of GFP neurons. (C) Mixed hippocampal cultures were transfected at 21 DIV with GFP (C1,E1 and insets C1',E1') or DA-GFP (D1,F1 and insets D1',F1'), and 1 day after transfection, cells were double immunostained for synaptophysin (C2,D2) and the subunit β2,3 of GABAA receptors (C4,D4) or for Gad-65 (E2,F2) and vGlut1 (E4,F4). All the synaptic markers were located either on the dendritic shafts or on dendritic protrusions. Interestingly, we observed in DA neurons that several dendritic spines share the same presynaptic terminal (see arrowheads in D3' vs C3' and F3' vs E3'). Scale bars: 10 µm in C1-F6 and 5 µm in insets in right column. (G) The average density of syn, β2,3, syn + β2,3, Gad-65 and vGlut1 clusters along dendrites of DA-GFP were not different from those of GFP neurons. Note that in addition to dendrites of transfected neurons, all synaptic markers also stained dendrites of nontransfected neurons.

 

Figure 5
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  		Fig. 5. Effects of DA-GFP overexpression on bassoon protein. Mixed hippocampal cultures were transfected at 21 DIV with GFP (A) or DA-GFP (B) and 1 day after transfection, cells were immunostained for bassoon (A',B'). (A'' and B'') Merge of panels A and A' and B and B', respectively. (A''' and B''') Composite of A'' and B'', and drawings showing selected objects obtained with ImageJ software from A' and B', respectively. (C,D) Histograms showing the density of bassoon clusters (C) and their number per spine (D) in GFP and DA-GFP neurons. ***P<0.0001, Student's t-test. Scale bar: 5 µm.

 

Figure 6
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  		Fig. 6. Effects of DA downregulation on network activity. Mixed hippocampal cultures of 21 DIV treated for 2 days in the absence (untreated) or the presence of 10 µM sense (S) or antisense (AS) oligonucleotides. Following treatment, the cells were either used for western blot analysis (A,B) or electrophysiology recordings (C,D). **P<0.001, Bonferroni's test. (C) Using whole-cell recordings, sPSCs were detected at –60 mV in sense- (black trace) and antisense-treated neurons (gray trace). (D) Histogram showing the sPSC average charge transfer in all recorded neurons.

 

Figure 7
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  		Fig. 7. Effects of DA downregulation on glutamate function. Mixed hippocampal cultures of 21 DIV treated for 2 days with 10 µM sense or antisense oligonucleotides, followed by electrophysiological recording. (A) Examples of mEPSCs recordings from DA sense (black trace) and antisense oligonucleotide-treated neurons (gray trace). (B,C) Cumulative probability plots of amplitude and frequency of mEPSCs in sense- and antisense-treated neurons. The average amplitude and frequency of mEPSCs in sense and antisense oligonucleotide-treated neurons are shown in insets. *P<0.05, K-S test. (D-F) Histograms showing the average rise time, decay time, and charge transfer of mEPSCs in oligonucleotide-treated neurons. ***P<0.001, U-test.

 

Figure 8
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  		Fig. 8. Effects of DA downregulation on GABA function. Mixed hippocampal cultures of 21 DIV treated for 2 days with 10 µM sense (S) or antisense (AS) DA oligonucleotides, followed by electrophysiological recording. (A) Examples of mIPSC recordings from sense (black trace) and antisense neurons (gray trace). (B,C) Cumulative probability plots of amplitude and frequency of mIPSCs in sense and antisense oligonucleotide-treated neurons. The average amplitude and frequency of mIPSCs in oligonucleotide-treated neurons are shown in insets. *P<0.05, **P<0.01, K-S test. (D-G) Histograms showing the average rise time, decay time, charge transfer and inhibition to excitation charge transfer ratio of mIPSCs in oligonucleotide-treated neurons. ***P<0.001, U-test.

 

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© The Company of Biologists Ltd 2009